Thymine DNA Glycosylase (TDG) is a DNA glycosylase involved in base excisionrepair, a DNA repair pathway that acts in a lesion-specific manner to correct individual damaged or altered bases. TDG preferentially catalyzes the removalof thymine and uracil paired with guanine, but is also active on5-flurouracil (5-FU) paired with adenine and guanine. There is a variant ofTDG found in 10% of the global population where residue Gly199 is mutated to Ser. Gly199 is responsible for stabilizing the flipped abasic nucleotide inthe active site pocket and mutation of this residue could affect the functionof this protein. Interestingly, we have shown that cells expressing G199Shave increased sensitivity to treatment with chemotherapeutic agent 5-FU compared to WT. Biochemical studies indicate G199S binds more tightly to anabasic product with a 4-fold lower Kd than WT. Additionally, expression ofG199S in cells leads to an increase in 5-FU-induced single-strand DNA breakscompared to WT, as well as persistent double-strand breaks after treatmentwith 5-FU and recovery in a drug-free media for up to 48hrs. Together, theseresults suggest G199S binds more tightly to its abasic product /in vivo/preventing efficient downstream processing of the BER intermediate andeventually leading to DNA break formation. I plan to further elucidate the cellular mechanism contributing to this increased sensitivity in hopes ofunderstanding the role of G199S in 5-FU-directed toxicity.